Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143Parker Institute for Cancer Immunotherapy, San Francisco, CA 94143Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan

Article Figures & Data

Figures

NK cells in NKp46-CreERT2 Tg mice carrying the Rosa26-tdTomato allele or the Rosa26-YFP alleles express tdTomato or YFP after tamoxifen administration. (A) Schematic representation of the strategy used to generate NKp46-CreERT2 Tg mice. An IRES-CreERT2 cassette was inserted into the 3′ untranslated region of exon 7 of the mouse Ncr1 gene on mouse chromosome 7 in a C57BL/6 BAC. (B–D) NKp46-CreERT2 Tg mice with a heterozygous Rosa26-tdTomato allele were treated with tamoxifen for 5 consecutive days and immune cells were analyzed after the last tamoxifen administration. (B) Expression of tdTomato in NK cells (DX5+NKp46+TCRβ−B220−); T cells (NKp46−TCRβ+B220−); B cells (NKp46−TCRβ−B220+); and non-NK, non-T, and non-B cells (NKp46−TCRβ−B220−, predominantly myeloid cells) in the spleen. (C) Expression of tdTomato in NK precursor cells (TCRβ−CD19−CD11b−DX5−NKp46−CD122+) and immature (CD11b−CD27+), intermediate (CD11b+CD27+), and mature (CD11b+CD27+) NK cells (TCRβ−CD19−DX5+NKp46+) in the bone marrow. (D) Expression of tdTomato in NK cells (TCRβ−CD19−DX5+NKp46+CD122+), liver-resident NK cells (TCRβ−CD19−DX5−NKp46+CD122+), ILC1 (TCRβ−CD19−DX5−NKp46+CD11b−CD27+CD122low), and NKp46+ ILC3 (TCRβ−CD19−DX5−NKp46+CD11b−CD27−CD122low that are potentially ILC3) in the liver. Data are representative of two to three experiments (n = 2 in each experiment). (E and F) NKp46-CreERT2 Tg mice with a heterozygous Rosa26-YFP allele (E and F) and homozygous Rosa26-YFP alleles (F) were treated with tamoxifen for 5 d, and then splenocytes were analyzed. (E) Expression of YFP in NK cells; T cells; B cells; and non-NK, non-T, and non-B cells (predominantly myeloid cells) in the spleen. (F) Expression of YFP by NK cells in the spleen. Data are representative of more than five experiments (n = 2–6 in each experiment). Bold and thin lines represent cells in mice treated with tamoxifen and cells in untreated mice, respectively.

IL-12 is required for the optimal differentiation of memory NK cells and cytokine-activated NK cells. NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated daily with tamoxifen on days 0–4 and infected with MCMV on day 0. Mice were injected with 200 µg control Ig or anti–IL-12 neutralizing mAb on the day before infection and on day 3 and 6 p.i. The percentages of YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the spleens on day 26 p.i. are shown. Data are pooled from two experiments (n = 4–5 in each group). *, P < 0.01; **, P < 0.005 versus control Ig. P-values were calculated by a Student’s t test. Error bars show SEM.

Cytokine-activated NK cells preferentially persist in an MCMV-free environment. (A) NKp46-CreERT2 Tg mice with a heterozygous Rosa26-tdTomato allele or homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with MCMV on day 0. On day 27 after treatment with tamoxifen, tdTomato+ NK cells from naive uninfected mice were mixed with YFP+ NK cells from naive uninfected mice and with MCMV-primed YFP+ NK cells from infected mice, and then adoptively transferred into Rag1-deficient B6 mice. Donor NK cells were analyzed on day 5 after the transfer. (B) Expression of Ly49H and KLRG1 on donor tdTomato+ NK cells and YFP+ NK cells before transfer and in the spleens of Rag1-deficient recipient mice on day 5 after transfer. Splenocytes on day 5 after transfer were fixed and permeabilized for staining of Ki67. Thus, the fluorescence of tdTomato and YFP decreases as compared with before transfer. Data are representative of two experiments (n = 3–4 in each experiment). (C) The number of donor NK cells in the spleens of Rag1-deficient recipient mice on day 5 after transfer. The y axis represents the number of donor NK cells detected in the spleens of Rag1-deficient mice on day 5 compared with the number of donor NK cells adoptively transferred into Rag1-defiicent mice. *, P < 0.01 versus memory YFP+ cells. (D) The percentages of Ki67+ donor NK cells on day 5. Data were pooled from two experiments (n = 6–7 in each group). *, P < 0.05 versus naive tdTomato+ cells and naive YFP+ cells. (E–G) NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with MCMV on day 0. On day 27 after tamoxifen treatment, YFP+ NK cells isolated from naive uninfected mice and MCMV-primed YFP+ NK cells were adoptively transferred into Rag1-deficient B6 mice. Donor NK cells were analyzed on day 5 after the transfer. (E) Expression of Bcl-2 in donor NK cells on day 5. Bold solid lines, bold dashed lines, and thin lines represent memory NK cells, cytokine-activated NK cells, and naive NK cells, respectively. A filled histogram represents staining with an isotype-matched control Ig. Data were representative of two experiments (n = 2–3 in each experiment). MFI of Bcl-2 staining in NK cells is shown in F. (G) The percentages of Annexin V+ donor NK cells on day 5. Data were pooled from two experiments (n = 5 in each group). *, P < 0.05 versus memory cells. (H–K) NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with MCMV on day 0. (H and I) On day 25 after tamoxifen treatment, YFP+ NK cells from naive uninfected mice or MCMV-primed YFP+ NK cells were mixed with CD45.1+ WT B6 NK cells from naive uninfected mice, labeled with CellTrace Violet, and cultured in the presence of 2 or 10 ng/ml IL-15 for 4 d. (H) Cell divisions of NK cells on day 4. Filled histograms represent naive YFP+ NK cells. Bold and thin lines represent YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells, respectively. The number of cell divisions of NK cells was quantified in I. Data are representative of two experiments (n = 3–6 in each experiment). *, P < 0.05 versus memory cells. (J) Expression of CD122 and CD132 on YFP+ NK cells in the spleens of naive uninfected mice and YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the spleens of infected mice on days 25–27 p.i. Bold solid lines, bold dashed lines, and thin lines represent memory NK cells, cytokine-activated NK cells, and naive NK cells, respectively. Filled histograms represent staining with an isotype-matched control Ig. Data are representative of more than three experiments (n = 2–3 in each experiment). MFI of CD122 and CD132 staining of NK cells are shown in K. Data were pooled from three experiments (n = 6–7 in each group). *, P < 0.005 versus naive cells. P-values were calculated by a Student’s t test. Error bars show SEM.

Memory NK cells exert augmented antitumor activity. NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with WT MCMV on day 0. (A and B) Degranulation (A) and IFN-γ production (B) of naive YFP+ NK cells from naive uninfected mice and YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells from infected mice after culture in the presence of IL-12 and IL-18, stimulation with anti-NK1.1 mAb, and co-culture with RMA transfectants expressing m157 or Rae1γ. Data are representative of three experiments (n = 3–4 in each experiment). *, P < 0.05. (C) Splenic YFP+ NK cells from naive uninfected mice, and YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells from infected mice were isolated on day 26 p.i. 15,000 YFP+ NK cells were transferred into T cell–depleted DAP10 and DAP12-double deficient recipient B6 mice. CellTrace Violet–labeled RMA-Rae1γ transfectants and RMA-S cells were mixed with RMA cells at a 1:1:1 (105 of each cell line) ratio, and injected intraperitoneally into these recipient mice on the day of NK cell transfer. An aliquot of the mixed tumor cells was cultured as a control. Proportions of these tumor cells (RMA, H-2Kb+ CellTrace Violet−; RMA-Rae1γ transfectants, H-2Kb+ CellTrace Violet+; and RMA-S cells, H-2Kb− CellTrace Violet+) in the peritoneal cavity were analyzed at 48 h after the injection. Data are representative of two experiments (n = 3 in each experiment). (D) NKG2D-mediated cytotoxicity against RMA-Rae1γ transfectants was quantified as the number of RMA-Rae1γ transfectants compared with the number of RMA cells detected in the peritoneal cavity at 48 h after injection. The y axis represents the clearance of RMA-Rae1γ transfectants in the peritoneal cavity of recipient mice receiving donor NK cells normalized to that of recipient mice not receiving donor NK cells expressed as a relative quantity. Data were pooled from two experiments (n = 6 in each group). *, P < 0.01 versus memory cells. P-values were calculated by a Student’s t test. Error bars show SEM.